Heterosis, or plant hybrid vigor, is a phenomenon where hybrid plants display superior phenotypes compared to either of its inbred parent lines. Hybrid vigor was discovered in maize breeding nearly a century ago, and has subsequently been found to occur in many crop species (Duvick D N 2001. Nat Rev Gent 2, 69-74). A large portion of the dramatic increase in agricultural output during the last half of the twentieth century has been attributed to the development and use of hybrid seed varieties in core crops including, for example, corn (maize), wheat, sorghum, sunflower, alfalfa and canola.
For maize, it is estimated that heterosis increases crop yields by at least 15%, which, in combination with modern higher yielding inbred lines and improved agronomic techniques, has resulted in a steady linear increase in performance. At the end of the last century, it was estimated that 65% of maize production worldwide was hybrid-based, with other crops, such as sorghum and sunflower, showing similar numbers. Taken together, increased yield advantages due to hybrids range between 15-50%, depending on the crop (Duvick D N 1999. In: The genetics and exploitation of heterosis in crops, J G Coors and S Pandey, eds. Madison: American Society of Agronomy, Inc. and Crop Science Society of America Inc. pp. 19-29).
With such great yield benefits, it is no surprise that the breeding of food and future biofuel crops is based on developing hybrid plants; yet the principles governing heterosis are still not understood. Efforts to decipher the genetic and molecular bases of heterosis so that its power can be harnessed and utilized more efficiently have so far proven unsuccessful. Over the years, crop plants have provided the genetic resources to study heterosis because parental inbred lines have been artificially selected for maximum hybrid combining ability, and the creation of structured genetic populations allows for robust quantitative phenotyping. Indeed, much of the knowledge on heterosis comes from classic genetic studies on maize, during which the fundamental hypotheses for heterosis were defined, involving genome-wide dominance complementation as well as locus-specific, or single gene, overdominant (ODO) effects.
Attempts to refine heterosis into genetic components began a decade ago with the development of molecular markers. Subsequent quantitative trait loci (QTL) mapping in rice and maize addressed the classical models by breaking down heterosis into “Mendelian” factors and assessing their modes of inheritance (Stuber C W et al., 1992. Genetics 132, 823-839; Xiao J et al., 1995. Genetic 140, 745-754; Luo et al., 2001. Genetics 158, 1755-1771; Hua J et al., 2003. PNAS 100, 2574-2579). The evidence suggested that both dominance and overdominance (ODO) have a role in heterosis, with some involvement of epistasis, although the relative contribution of each of these mechanisms was unclear.
Little progress has been made towards the identification of genes resulting in heterotic loci, largely because of the complexity of the phenotypic interactions that define heterosis and the efforts required for QTL cloning. Despite the availability of genomic infrastructure for a wide range of plant models and suitable genetic populations, such as introgression line (IL) populations, mapping and cloning QTL with heterotic effects remains extremely challenging, and single genes causing heterosis for crop yield have still not been identified. This is primarily because most conventional QTL studies begin with the goal of mapping multiple loci for a defined phenotype, and those QTL that have been already cloned generally involve loci with significantly large effects and high heritabilities. Heterosis, in contrast, bears little similarity to previously cloned QTL as its manifestation is based on complex interactions between phenotypic components throughout development, each with its own mode of heritance, and a dynamic influence of the environment (Lippman Z B and Zamir D, TIG, 2007, 23, 60-6). This is generally referred to as “multiplicative” or “geometric” heterosis. Therefore, it can be assumed that mapping heterotic QTL is equivalent to mapping multiple, perhaps genetically unrelated traits simultaneously. This integration of traits makes the heritability of heterosis relatively low compared to more discrete phenotypes, such as fruit weight or sugar content (Frary A et al., 2000. Science 289, 85-88; Fridman et al., 2004. Science 305, 1786-1789). This complexity is highly relevant to the classic heterosis phenotype of total yield.
The cloning of heterotic genes may require additional genomic and nearly isogenic resources. Furthermore, while there are examples of chromosomal regions that may carry genes associated with heterotic yield due to pseudo-ODO (Eshed Y and Zamir D 1995. Genetics 141, 1147-1162; Graham G I et al., 1997. Crop Science 37, 1601-1610), there are no known true-ODO genes based on single gene effects. Recent data from tomato ILs provide indirect support for true-ODO (Semel Y et al., 2006. Proc Natl Acad Sci USA 103, 12981-12986). Using a phenomics approach as described above, ODO-QTL were found to be preferentially associated with traits for increased reproductive fitness, such as yield, whereas dominant, recessive and additive QTL were dispersed throughout the phenotypic categories, including non-reproductive traits. This selective association suggests that pseudo-ODO is unlikely to explain IL heterosis, as the expected overdominant-QTL for the non-reproductive traits, assuming random distribution of increasing dominant and decreasing recessive QTL throughout the genome, was higher compared to what was actually found. A heterosis study in Arabidopsis arrived at a similar conclusion on the basis of fewer phenotypes (Mitchell-Olds T., 1995. Genetics 140, 1105-1109).
Since heterosis is a genome-wide phenomenon, it has been speculated that its molecular mechanisms involve global changes in gene and protein expression. Recently, a comprehensive analysis on gene expression in inbreds and hybrids of B73 and Mo17 maize lines was carried out using microarrays. With nearly 14,000 genes assayed from seedling tissue, it was concluded that overdominant gene expression patterns could contribute to heterosis, acting along with all other mechanisms of gene expression, including additivity and dominance. Interestingly, a nearly identical microarray study in maize using the same parental inbred lines and hybrids came to a different conclusion where gene expression is mainly additive in heterotic hybrids, with almost no examples of ODO. The different conclusions may be the result of inherent technical issues associated with microarrays, including the source of the microarray platforms, different statistical thresholds and the like.
Mapping expression-QTL (eQTL) and analyzing the association between eQTL and phenotypic QTL has been also suggested as a tool to elucidate the molecular basis of heterosis. However, taking this approach may lead to obscured results based on the assumption that gene expression overdominance explains growth and morphological overdominance when, in fact, expression overdominance is simply one of many molecular phenotypes. It is more prudent to assume that the molecular mechanism underlying expression overdominance is independent from that of morphological phenotypic overdominance. An illustration of this concept comes from mis-expressed genes in inter-specific hybrids of Drosophila, which are the result of cis-trans compensatory evolution where the interaction of the trans-regulatory elements from one species with the cis-regulatory elements of the other is responsible for the dysregulation in the hybrids. Importantly, expression studies in other diploid and polyploid plants, as well as animals and yeast, using a variety of techniques show that non-additive gene expression in hybrids is a common occurrence. The fact that the majority of these studies are outside the context of heterosis suggests that there is no obvious link between global expression changes due to heterozygosity and hybrid vigor. In fact, gene expression changes in hybrids may be downstream molecular responses driven by heterotic growth effects and the genes controlling same (Schauer N et al., 2006. Nat Biotechnol 24, 447-454).
There is an ongoing attempt to develop methods and means for selecting crop plants showing heterosis using molecular and computer modeling techniques.
For example, International Patent Application Publication No. WO 00/42838 discloses methods of correlating molecular profile information with heterosis. The molecular profiling includes RNA or protein expression in a tissue of a plant, enabling the prediction of the heterosis level the plant will display if tested for a heterotic trait such as yield. That invention further discloses that selection for dominant, additive, or under/overdominant molecular profiling markers as well as selection for the number of expression products in an expression profile provides for improved heterosis. Methods of identifying and cloning nucleic acids linked to heterotic traits and for identifying parentage by consideration of expression profiles are also provided.
International Patent Application Publication No. WO 03/050748 discloses methods, computer program products, and systems for statistical analyzes of differential gene expression from hybrid offspring and their inbred parents, and the identification of genes that play a role in heterosis.
U.S. Patent Application Publication No. 2009/0170712 discloses a method for prediction of the degree of heterotic phenotypes in plants. Structural variation analyses of the genome and, in some examples, copy number variation are used to predict the degree of a heterotic phenotype in plants. In some methods copy number variation is detected using competitive genomic hybridization arrays. Methods for optimizing the arrays are also disclosed, together with kits for producing such arrays, as well as hybrid plants selected for development based on the predicted results.
U.S. Pat. No. 7,084,320 discloses methods and means for determining parent inbred plant lines with good combining ability, for determining good combinations of parent inbred plant lines capable of yielding hybrid lines with high heterosis, and further for determining the agronomical performance of different plant lines, which can be performed in vitro by determining the electron flow in the mitochondria under control and stress conditions.
However, as described hereinabove, the ability to predict and identify genes governing heterosis that can be used in crop breeding is limited, and there is an unmet need to, and would be highly advantageous to have readily defined parent inbred lines for breeding plants with hybrid vigor.